Trehalose signal promotes flowering through MlTPS1-MlNFYC9 regulation module in Magnolia liliiflora Desr

The trehalose pathway primarily regulates flowering in plants through T6P (Trehalose-6-Phosphate) and TPS1 (TREHALOSE-6-PHOSPHATE SYNTHASE 1), although the specific regulatory mechanism remains uncertain. This study utilized full-length transcriptome and RNA-seq data to identify key genes responding to trehalose signal and impacting flowering in Magnolia liliiflora 'Hongyuanbao'. Analysis revealed 90, 438, 1,544, and 667 differentially expressed genes (DEGs) throughout the four flower bud differentiation stages (S1∼S4). GO and KEGG enrichment analyses indicated enrichment of DEGs from S1 to S4 in photoperiod and circadian rhythm pathways. Weighted gene co-expression network analysis (WGCNA) identified three significant co-expression modules, with significant correlations between the MEbrown and MEbrown modules. DEGs in the MEbrown module primarily participated in light response and carbon metabolism pathways, while DEGs in the MEblue module were primarily associated with stress response and sugar metabolism pathways. Additionally, protein-protein interaction (PPI) network analysis of flowering and sugar-related DEGs from both modules identified MlTPS1 as a central protein in the interaction network. Yeast two-hybrid (Y2H) analysis combined with bimolecular fluorescence complementation (BiFC) assays confirmed that MlTPS1 interacts with the flowering-promoting factor MlNFYC9 (NUCLEAR TRANSCRIPTION FACTOR Y SUBUNIT C-9) in the cytoplasm. Functional verification and expression analysis results indicated that MlNFYC9 significantly promotes the flowering process and exhibits prominent expression in the flower bud and early flower bud differentiation stages. This study elucidated the molecular mechanism by which 'Hongyuanbao' augments reproductive development under trehalose signal, providing a theoretical basis for refining flowering time regulation in Magnolia species.